The lettuce aphid (Nasonovia ribisnigri (Mosley)) is a major pest occurring in lettuce worldwide. The problem started to be severe for lettuce production in the 1970's in North Western Europe and spread rapidly all across Europe. Then, in the 1980's, the aphid was detected in Canada. Later on, the problem was reported in the USA (California and Arizona). More recently, the lettuce aphid was found in New Zealand and Australia.
Lettuce aphids can colonize lettuce plants at any plant stage and feed preferably from younger leaves. Large amount of aphids on the plant are able to reduce plant growth and deform the shape of the head so that the lettuce heads are then not marketable. The presence of high amounts of aphids in lettuce heads is a reason for retailers to refuse to buy lettuce from growers. At young plant stage it is possible to control the lettuce aphid using insecticide. Several products were reported to be efficient in controlling aphid populations. However, resistance to chemicals were reported in some aphid populations. Moreover, at older developmental stages it is not possible to control aphids using insecticides, as chemical products cannot enter into the lettuce head.
Since 2007, two biotypes of lettuce aphid have been known in Europe, which were designated biotype Nr:0 and Nr:1. Complete and partial resistance against Nasonovia ribisnigri biotype Nr:0 were found in Lactuca virosa, a wild relative of lettuce (Eenink and Dieleman, Euphytica 32(3), 691-695 (1982)). The complete resistance was due to a single dominant gene, termed the Nr gene. The Nr gene was transferred from L. virosa accession IVT280 into cultivated L. sativa and was highly effective (Arend et al. 1999, Eucarpia Leafy Vegetables '99. Palacky University, Olomouc, Czech Republic, p 149-157).
However, breeders experienced that the release of varieties resistant to lettuce aphid was not straightforward. The Nr resistance gene was found tightly linked to recessive genes conferring strong negative side-effects. Plants homozygous for the Nr gene showed a reduced growth, a lighter green colour and accelerated degradation of chlorophyll in the older leaves. This negative phenotype was also referred to as the “Compact Growth and Rapid Ageing” phenotype or “CRA phenotype” and it was possible to find recombinant lettuce plants in which the Nr gene was present in homozygous form, but in which the CRA phenotype was not expressed (see, e.g., EP 0921720 B1). These recombinant plants, in which a recombination event (i.e. meiotic crossing-over) had taken place between the Nr gene and the linked recessive genes, served as the source of the Nr resistance gene that was not linked to the negative side-effect phenotype.
The Nr resistance gene from IVT280 (CGN04683) is widely used in commercial lettuce cultivars, such as cultivars ‘Barcelona’, ‘Mafalda’ (both Nunhems B.V.) and many others.
Other sources of biotype Nr:0 resistance are still being sought, as the wide-scale use of a single resistance gene faces the threat of resistance breakdown. Genes which have different resistance mechanisms can be effectively employed in such circumstances. For example new biotype Nr:0 resistance genes were found in an L. serriola accessions PI 491093 and in an L. virosa accession PI 274378 (Mc Creight 2008 HortScience 43:1355-1358; McCreight and Liu (2012), HortScience 47(2):179-184). The resistance found in PI274378 was found to be complete and allelic to the Nr gene from IVT280. In PI49093 partial resistance was found, and the authors proposed to designate this resistance allele Nr0P (in contrast to Nr0C for the complete resistance allele found in PI274378). They suggested using this partial resistance allele in areas where the complete resistance allele has not yet been widely employed in order to delay or prevent emergence of aphid biotypes that overcome Nr resistance.
Also resistance against Nasonovia ribisnigri biotype Nr:1 is sought after. Different resistance genes and resistance mechanisms are also desired regarding Nr:1, to prolong the use of resistance genes. When large scale use is made of one resistance gene, which has a certain resistance mechanism, the chances are high that resistance will be overcome by the aphid population, as happened in 2007 for the Nr gene in Europe, when the new Nasonovia ribisnigri biotype Nr: 1 appeared. Thus, the current situation is that the aphid biotype Nr:0 can still be controlled by the single Nr gene derived from the IVT L. virosa accession (for example in the USA, where biotype Nr:1 does not yet occur), but that this gene is ineffective against aphid biotype Nr:1 found in Europe. The Nr: 1 biotype was first found only in central Europe, but is now spreading to other areas and in 2010 has also been found in fields in Spain (Cid et al. 2012, Arthropod-Plant Interactions 6: 655-669).
Several publications describe sources which are suggested to contain a resistance gene against lettuce aphid biotype Nr:1. For example three accessions (CGN13361, CGN16266, CGN16272) were described to be resistant against both aphid biotypes Nr:0 and Nr:1 (Anonymous, 4, Nov. 2008, IP.COM document 000176078) and were suggested to be used in breeding for combined Nr:0 and Nr:1 resistant lettuce. In this article L. virosa accessions CGN16272 and CGN16266 are said to be used in backcrossing programs with cultivar Daguan (Syngenta) and cultivar Funly (Syngenta), respectively (both these cultivars lack Nasonovia resistance genes), and the offspring are said to show a resistance similar to the resistance of the donor accessions. Also markers for the resistance gene of CGN16272 are said to be developed from crosses of CGN16272 with cultivar Cobham Green (Anonymous, 4, Nov. 2008, supra). These three accessions were also analyzed in Cid et al. (2012, supra) and were found to comprise high Nr:0 resistance but only partial biotype Nr:1 resistance.
Cid et al. (2012, supra) also identified three L. virosa accessions with some resistance against both biotypes Nr:1 and Nr:0, namely CGN16274, CGN21399 and CGN05148. In this study, the authors aim was to find resistance against both lettuce aphid biotypes, Nr: 0 and Nr: 1, in single wild Lactuca accession. However, aphids of biotype Nr:1 are still able to feed and reproduce on these wild accessions, albeit to a lower extent than on the susceptible controls (see FIG. 4).
WO2011/058192 reports L. serriola 10G.913571 as being resistant against biotype Nr:1, although no data are provided to substantiate this claim and no indication of the level of resistance and methods to determine this is given.
WO2012/066008 and WO2012/065629 also report Nr: 1 resistance from an L. serriola accession to be transferred into a bulk seed sample designated 10G.913569. Again, no data are provided and no indication on the resistance level and methods to determine resistance is given.
Thus, in the prior art, such as Anonymous 2008, above, and Cid et al. 2012, above, only a few wild accessions are identified on which the amount of Nr: 1 aphids is reduced to some extent and no genetic basis is provided.
There remains a need for identifying genes which can confer resistance against biotype Nr:1 in order to develop cultivated lettuce comprising Nr:1 resistance. The instant inventors looked for accessions which were thought to be susceptible to aphids of biotype Nr:0, in order to identify (new) resistance gene(s) against biotype Nr: 1 in these accessions. In addition, they also looked for the identification of genes which can confer both free-choice and non-choice resistance. They found a L. virosa accession (of which a representative sample of seeds was deposited under NCIMB42086) comprising high levels of resistance against biotype Nr:1, both under free choice and non-choice conditions, and decided to try to map the resistance, in order to identify how many and which L. virosa genome regions are responsible for conferring Nr:1 resistance.
When trying to map the resistance, the inventors encountered severe problems in creating a population of plants useable for QTL mapping (i.e. a mapping population which consists of individuals that have undergone chromosomal meiotic recombination between the L. sativa and L. virosa genomes). The reason is likely that the chromosomes of L. virosa and L. sativa, which are two different species, are quite different, leading to crossing barriers and infertility, as well as potential problems during meiosis and crossing over. No useable F2 populations could be generated, and only after many crosses with various recurrent parents the inventors succeeded in generating large enough backcross families which could be used in mapping studies. These mapping populations were also not easy to analyze using molecular markers and phenotyping, i.e. it was quite surprising that the inventors managed to generate a genetic map with SNP markers that are polymorphic between the recurrent parent and the L. virosa accession, and were also able to map Nasonovia Nr:1 resistance onto that map.
Surprisingly, in the initial QTL mapping study (using a BC1 population) they did not find a single gene, but three genomic regions (of which only two were later also found in a different backcross population) on two different chromosomes of L. virosa which contribute to the Nr:1 resistance. Both controlled environment inoculations (free choice and non-choice) and field data (also free choice and non-choice) showed high levels of Nr:1 resistance, against three geographically distinct Nr:1 biotypes (Germany, France and Spain). In fact, in field evaluations in Spain (Murcia) semi-adult and adult plants of the accession NCIMB42086 did not have any Nr:1 aphids on their leaves in both free-choice and non-choice tests.
In the later mapping study, two of the QTLs (QTL6.1 and QTL7.1) were found again and the QTL region could be narrowed down. This second mapping study does not invalidate the results of the first study, and all three QTLs are encompassed herein.
It is an object of the invention to provide three QTLs (designated QTL6.1, QTL7.1 and/or QTL7.2) from L. virosa which can be used to generate cultivated lettuce plants comprising resistance against biotype Nr:1.
It is also an object of the invention to provide cultivated lettuce plants comprising one or two or three QTLs (QTL6.1 and/or QTL7.1 and/or QTL7.2) introgressed from a wild lettuce, such as L. virosa, into the L. sativa genome, whereby the introgressions confer resistance against biotype Nr: 1.
Thus, different cultivated lettuce plants are encompassed herein: a) cultivated lettuce plants comprising only one QTL conferring Nr:1 resistance, selected from QTL6.1, QTL7.1 and QTL7.2; b) cultivated lettuce plants comprising two QTLs conferring Nr:1 resistance selected from QTL6.1 and QTL7.1 and QTL7.2 (in one aspect a plant comprising both QTL6.1 and QTL7.1 is a specific embodiment); c) cultivated lettuce plants comprising three QTLs conferring Nr:1 resistance selected from QTL6.1, QTL7.1 and QTL7.2.
It is also an object of the invention to provide cultivated lettuce plants comprising one or two QTLs selected from QTL6.1 and QTL7.1 introgressed from a wild lettuce, such as L. virosa, into the L. sativa genome, whereby the introgressions confer resistance against biotype Nr: 1.
In one aspect the QTLs are obtainable from (are as in) seeds deposited under accession number NCIMB42086. The introgression fragment(s) comprising the QTL(s) is/are detectable by a molecular marker assay which detects at least 1, 2, 3, 4, or more markers. In another aspect the QTLs are obtainable from (are as in) other Nr:1 resistant wild lettuce accessions, especially in L. virosa accessions, whereby the introgression fragment(s) is/are detectable by a molecular marker assay which detects at least 1, 2, 3, 4, or more (i.e. 5, 6, 7, 8, 9, 10, 11, 12, or more) markers disclosed herein. In one aspect the L. virosa accession is one of two types of accessions, and the introgression fragment comprises a L. virosa accession specific SNP marker (named VSP for Virosa Specific), selected from VSP1 and VSP2, both specific for one L. virosa accession and VSP3 and VSP4, both specific for another L. virosa accession.
Despite problems in interspecific QTL mapping mentioned above, like infertility, segregation distortion, etc., the originally found QTL regions (which were originally mapped to a physical region spanning 60 to 240 Mb on chromosome 6; and 170 to 235 Mb on chromosome 7 for QTL7.1; and 70 to 150 Mb for QTL 7.2) could be mapped to a region spanning 77 Mb to 161 Mb on chromosome 6 (comprising QTL6.1) and 203 Mb to 219 Mb on chromosome 7 comprising QTL7.1.
Thus, in one aspect a cultivated Lactuca sativa plant is provided comprising an introgression fragment on chromosome 6 (comprising QTL6.1) and/or on chromosome 7 (comprising QTL7.1), each in homozygous or heterozygous form, wherein said introgression fragment confers resistance against Nasonovia ribisnigri biotype 1 (Nr:1). In one aspect the introgression fragment comprises all or part of the region starting at 77 Mb on chromosome 6 and ending at 161 Mb on chromosome 6 and/or the introgression fragment comprises all or part of the region starting at 203 Mb on chromosome 7 and ending at 219 Mb on chromosome 7. See e.g. FIG. 3B, showing L. sativa chromosomes 6 and 7, where the gray bars illustrate the introgression fragments from a wild Nr:1 resistant accession, such as a L. virosa accession (e.g. NCIMB42086) comprising the resistance conferring QTLs QTL6.1 and QTL7.1, or variants thereof. In one aspect an introgression fragment comprising QTL7.2 may also optionally be present in the cultivated lettuce plant.
It is understood that a smaller introgression fragment (i.e. comprising a resistance conferring part of the above mentioned region spanning 77 Mb to 161 Mb of chromosome 6) which retains the QTL6.1 (or variant) may be a fragment having a size of 80 Mb, 70 Mb, 60 Mb, 50 Mb, 40 Mb, 30 Mb, 20 Mb, 10 Mb, 5 Mb, 2.5 Mb, 2 Mb, 1 Mb, 0.5 Mb, 100 kb, 50 kb or less and comprise the QTL6.1 or a variant thereof. In one aspect the part is at least 5 kb, 10 kb, 20 kb in size, or more.
It is further understood that a smaller introgression fragment (i.e. comprising a resistance conferring part of the above mentioned region spanning 203 Mb to 219 Mb of chromosome 7) which retains the QTL7.1 (or variant) may be a fragment having a size of 15 Mb, 10 Mb, 5 Mb, 2.5 Mb, 2 Mb, 1 Mb, 0.5 Mb, 100 kb, 50 kb or less and comprise the QTL7.1 or a variant thereof. In one aspect the part is at least 5 kb, 10 kb, 20 kb in size, or more.
In one aspect, the introgression fragment on chromosome 6 is detectable by a molecular marker assay which detects at least one, preferably at least 2 or 3 or 4 or 5 (or more) of the markers selected from the group consisting of:                a) The CC or CT genotype for the Single Nucleotide Polymorphism marker SNP1.23 in SEQ ID NO: 23 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 23);        b) the CC or CT genotype for the Single Nucleotide Polymorphism marker SNP_02 in SEQ ID NO: 2 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 2);        c) the TT or CT genotype for the Single Nucleotide Polymorphism marker SNP2.24 in SEQ ID NO: 24 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 24);        the AA or AC genotype for the Single Nucleotide Polymorphism marker SNP_03 in SEQ ID NO: 3 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 3);        d) any wild lettuce genome specific marker, especially L. virosa-genome specific marker, located physically in-between SNP1.23 and SNP_03 (e.g. in-between SNP1.23 and SNP2.24, SNP1.23 and SNP_02); or in between SNP_02 and SNP_03 (e.g. in-between SNP_02 and SNP2.24); or in between SNP2.24 and SNP_03;        e) any wild lettuce genome specific marker especially L. virosa-genome specific marker, located within a distance of 10 Mb, preferably within 5 Mb, of any marker selected from SNP1.23, SNP_02, SNP2.24, or SNP_03.        
Optionally, in one aspect, the introgression fragment comprises (and is detectable by) a L. virosa accession specific marker selected from the GG or GT genotype for the Single Nucleotide Polymorphism marker VSP1 in SEQ ID NO: 26 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 26) and the AA or AC genotype for the Single Nucleotide Polymorphism marker VSP3 in SEQ ID NO: 27 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 27). Using the SNP markers VSP1 and VSP3 the introgression fragments comprising QTL6.1 from two different L. virosa type accessions can be distinguished.
In another aspect, the introgression fragment at the far end of chromosome 7 comprising QTL 7.1 (at a physical position between 203 Mb and 219 Mb of chromosome 7) is detectable by a molecular marker assay which detects at least one, preferably at least 2 or 3 or 4 or 5 (or more) of the markers selected from the group consisting of:                a. the TT or TC genotype for the Single Nucleotide Polymorphism marker SNP_17 in SEQ ID NO: 17 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 17);        b. the TT or TC genotype for the Single Nucleotide Polymorphism marker SNP17.25 in SEQ ID NO: 25 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 25);        c. the GG or GC genotype for the Single Nucleotide Polymorphism marker SNP_18 in SEQ ID NO: 18 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 18);        d. the GG or GA genotype for the Single Nucleotide Polymorphism marker SNP_19 in SEQ ID NO: 19 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 19);        e. any wild lettuce genome specific marker, especially L. virosa-genome specific marker, located physically in-between SNP_17 and SNP_19 (e.g. in-between SNP_17 and SNP_18, in between SNP_17 and SNP17.25; or in between SNP17.25 and SNP_19, or in between SNP17.25 and SNP_18, or in between SNP_18 and SNP_19);        f. any wild lettuce genome specific marker especially L. virosa-genome specific marker, located within a distance of 12 Mb, 10 Mb, preferably within 5 Mb, of any marker selected from SNP_17, SNP_17.25, SNP_18 and SNP_19.        
Optionally, in one aspect, the introgression fragment comprises (and is detectable by) a L. virosa accession specific marker selected from the CC or AC genotype for the Single Nucleotide Polymorphism marker VSP2 in SEQ ID NO: 28 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 28) and the GG or GA genotype for the Single Nucleotide Polymorphism marker VSP4 in SEQ ID NO: 29 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 29). Using the SNP markers VSP2 and VSP4 the introgression fragments comprising QTL7.1 from two different types L. virosa accessions can be distinguished.
When referring to the introgression fragment being “detectable by a molecular marker assay which detects” one or more markers, this means that the introgression fragment comprises the resistance genotype of that marker.
In a further aspect, seeds, plants and plant parts or cultivated lettuce comprising an introgression fragment from a wild lettuce, such as from L. virosa, comprising QTL6.1 and/or QTL7.1 (and optionally QTL7.2) is provided, whereby the introgression fragment confers resistance against Nasonovia ribisnigri biotype Nr:1. In one aspect the introgression fragment is from L. virosa, especially from L. virosa accessions comprising Nr:1 resistance in both free choice and non-choice tests as described herein. In one aspect the introgression fragment is from accession NCIMB42086 or is obtainable from accession NCIMB42086, or progeny or descendants thereof.
In another aspect the introgression fragment is from a L. virosa accession which comprises the following L. virosa accession specific SNP markers: the GG genotype (homozygous) or GT genotype (heterozygous) at nucleotide 71 of SEQ ID NO: 26 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 26), named the VSP1 marker; and the CC genotype (homozygous) or AC genotype (heterozygous) at nucleotide 71 of SEQ ID NO: 28 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 28), named VSP2. VSP1 and VSP2 are found in Nr:1 resistant accessions, such as NCIMB42086.
In another aspect the introgression fragment is from a L. virosa accession which comprises the following L. virosa accession specific SNP markers: the AA genotype (homozygous) or AC genotype (heterozygous) at nucleotide 71 of SEQ ID NO: 27 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 27), named the VSP3 marker; and the GG genotype (homozygous) or AG genotype (heterozygous) at nucleotide 71 of SEQ ID NO: 29 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 29), named VSP4. VSP3 and VSP4 are found in other Nr: 1 resistant accessions.
Also methods for making and/or identifying and/or selecting cultivated lettuce plants comprising an introgression from wild lettuce, such as from L. virosa, on chromosome 6 (comprising QTL6.1) and/or chromosome 7 (comprising QTL 7.1 and/or QTL7.2) are provided, as are methods for transferring QTLs to different cultivated lettuce plant lines or varieties, especially to Nr:1 susceptible lettuce lines or varieties.